STAT proteins were originally discovered as latent cytoplasmic transcription factors that mediate cytokine and growth factor responses (1, 2). Seven members of the family, Stat1. Stat2. Stat3. Stat4. Stat5a and Stat5b, and Stat6, mediate several physiological effects including growth and differentiation, survival, development and inflammation. STATs are SH2 domain-containing proteins. Upon ligand binding to cytokine or growth factor receptors. STATs become phosphorylated on critical Tyr residue (Tyr705 for Stat3) by growth factor receptors, cytoplasmic Janus kinases (Jaks) or Src family kinases. Two phosphorylated and activated STAT monomers dimerize through reciprocal pTyr-SH2 domain interactions, translocate to the nucleus, and bind to specific DNA-response elements of target genes, thereby inducing gene transcription (1, 2). In contrast to normal STAT signaling, many human solid and hematological tumors harbor aberrant Stat3 activity (3-8 for reviews).
Constitutive Stat3 activity mediates dysregulated growth and survival, angiogenesis, as well as suppresses the host's immune surveillance of the tumor, making constitutively-active Stat3 a critical molecular mediator of carcinogenesis and tumor progression.
Genetic and other molecular evidence reveals persistent Tyr phosphorylation of Stat3 is mediated by aberrant upstream Tyr kinases and shows cancer cell requirement for constitutively-active and dimerized Stat3 for tumor maintenance and progression. Thus, in numerous proof-of-concept studies (9-13), inhibition of Stat3 activation or disruption of dimerization induces cancer cell death and tumor regression. How aberrant Stat3 is regulated for meeting the tumor-specific requirements in malignant cells remains undefined. There have been no studies into defining the molecular details of how malignant cells regulate aberrant Stat3 and how this regulation changes upon Stat3 inhibition prior to the onset of phenotypic changes, although knowing these events will facilitate efforts in modulating aberrant Stat3 for managing human cancers. Small-molecule Stat3 inhibitors thus provide tools for probing the molecular dynamics of the cellular processing of Stat3 to understand Stat3's role as a signaling intermediate and a molecular mediator of the events leading to carcinogenesis and malignant progression.
The computational analysis of the interaction between the Stat3 SH2 domain-binding pTyr peptide sequence and the SH2 domain, per the X-ray crystal structure of Stat3b bound to DNA (14), can generate valuable information about key structural requirements for the Stat3:Stat3 dimer formation that will facilitate the design of effective small-molecules to disrupt the dimer. Such molecules can be used for therapeutic purposes and as tools for investigating the regulation of Stat3 protein. In the molecular modeling of the Stat3 pTyr-SH2 domain interaction, the peptidomimetic inhibitor, ISS 610 (10) derived from the Stat3 SH2 domain-binding pTyr (Y*) peptide, PY*LKTK (9), was used as a chemical probe for interrogating the Stat3:Stat3 dimer interface in order to derive non-peptide mimics.
Herein, we describe the design and characterization of a novel oxazole-based peptidomimetic, designated S3I-M2001, as a selective disruptor of Stat3:Stat3 dimerization and inhibitor of Stat3 activation. We also describe our study of the stability and intracellular processing of aberrant Stat3 within the context of the activity of S3I-M2001 as a Stat3 inhibitor.